TRIM5α recruits HDAC1 to p50 and Sp1 and promotes H3K9 deacetylation at the HIV-1 LTR

Tripartite motif-containing protein 5α (TRIM5α) is generally known to block the postentry events of HIV-1. Here, we report an uncharacterized role for TRIM5α in the maintenance of viral latency. Knockdown of TRIM5α potentiates the transcription of HIV-1 in multiple latency models, which is reversed by shRNA-resistant TRIM5α. TRIM5α suppresses TNFα-activated HIV-1 LTR-driven as well as NF-κB- and Sp1-driven gene expression, with the RING and B-box 2 domains being the essential determinants. Mechanistically, TRIM5α binds to and enhances the recruitment of histone deacetylase 1 (HDAC1) to NF-κB p50 and Sp1. ChIP‒qPCR analyses further reveal that the association of TRIM5α with HIV-1 LTR induces HDAC1 recruitment and local H3K9 deacetylation. Conserved suppression effects of TRIM5α orthologs from multiple species on both HIV-1 and endo-retroelement HERV-K LTR activities have also been demonstrated. These findings provide new insights into the molecular mechanisms by which proviral latency is initially established and activatable proviruses are resilenced by histone deacetylase recruitment.

The authors show that depletion of TRIM5 in human cells increases basal and TNF-induced transcription from an integrated HIV-1 LTR in T cell lines including Jurkat CD4+ cells, and HeLa cells. The effect was reverse by restoration of TRIM5 expression with a transgene, supporting a TRIM5-specific effect. The effect was mapped to the NF-kB and Sp1 sites in the HIV-1 LTR, but not interference with IkBa phosphorylation, and to the TRIM5 RING and B-Box domains. Mechanistically, TRIM5 promoted recruitment of HDAC1 to Sp1 and NF-kB proteins, and HDAC1 to the LTR, which was translated in a reduction in local H3K9 acetylation. TRIM5 itself was bound to HIV LTR-containing DNA. Repression of HERV-K was also observed. Figure 7 contains perhaps some of the more compelling data in this paper, showing TRIM5 association with viral DNA, which is a surprising finding not yet predicted by the current TRIM5 literature. However, this reviewer has several concerns regarding the presentation and interpretation of the data in the manuscript, as outlined below. Overall, some of the suppressive phenotypes are inconsistent, and the conclusions not always supported by the data as shown, in some cases misinterpreted. The Western blots are often difficult to interpret and therefore do not always support the conclusions reached.
An effect of TRIM5 on expression of integrated HIV provirus is surprising given its potent capsid binding activity and restriction of early steps of retrovirus infection. TRIM5 has not previously been implicated in this stage of the HIV lifecycle. If true, this would represent a significant advance for the field, however further data is required to support some of the claims made. Is there any evidence that TRIM5 expression correlates with HIV quiescence in vivo?
Major comments: General: Western blots: • Molecular weight indicators should be displayed on all western blot panels for it to be possible to interpret them. • Exposure -e.g. fig 4h tubulin, it's impossible to interpret as the exposure is too high. This is a recurrent concern.
How were TRIM5 mutants constructed -were these deletions, what were the domain boundaries? How was proper protein folding accounted for or is for example the ∆Box simply misfolded? If interference with higher order assembly or E3 ligase is required, single point mutants informed by domain structures are now known, for example B-Box R119E in human TRIM5 completely abolishes higher order assembly but not inherent E3 of the RING domain. In the RING domain, E11R or L19R selectively disable E2 binding and thus RING activity, without impacting on overall restriction of retroviral infection. Other mutants are available. Such mutants should be employed to narrow the effect on specific TRIM5 function, otherwise these deletion mutants might simply be non-functional generally and thus not informative. P6 line 120 -Query the interpretation of 1d. Jq1 is the only LRA that loses its phenotype in TRIM5 depleted cells, suggesting that TRIM5 repression and the target of Jq1 are mechanistically linked? Indeed, one model could be that TRIM5 suppresses transcription elongation, rendering JQ1 ineffective in the KD cell. The conclusion in the text 'suggesting that TRIM5 had no effect on transcription elongation' is not supported by the data. Pg 8 Line 155 -7c -the interpretation is questioned. The data to this reviewer suggests that LRAs (Latency reversing agents) and TRIM5 work independently, as the drugs work as effectively in WT or KD cells, but the base line is higher in the KD scenario.

Figure 3
Switched GFP for Luc -not clear why -so essentially similar data as presented in Figures 1-2.
What is the basis for switching between a stable LTR suppression assay as in Figures 1-2, and a transient LTR expression as employed here. In Figure 3, cotransfection of TRIM5 and LTR-HIV constructs mean that the regulation at the LTR is likely to be wholly distinct to the regulation measured in the chronically infected scenario. Some comment on this would be valuable.
3c -Interesting result. How is TRIM5 only able to antagonise TNF-induced transcription and not Tat driven elongation, when the latter requires transcription initiation and production of the TAR element to which Tat binds (in complex with CDK9 and Cyclin T1)? What is the logic for this experiment?
3d -we can't see the values in untreated conditions. Log scale would be clearer. How were these graphs normalised (if at all)?
3e -empty plasmid control is missing. How were these values normalised to one another? 3c-3e -why are the RLU units so different between these experiments?
In 3h-i, there seems to be a TNF-independent TRIM5 phenotype. In 3m-n, there seems to be a TNF-dependent TRIM5 phenotype. Would the authors comment on this? Both assays involved TRIM5 depletion.
The authors do not entertain the possibility of clonal artefact. Either multiple KO clones should be compared, or 1-2 clones should be reconstituted with TRIM5 to restore the parental phenotype. Otherwise, clone 10# could just be moderately more sensitive to TNF than the parental line. Same concern as above re: clonal effect.
4c -we can't see the black bars or any change between conditions, either separate panels or a log panel should be used, although this might alter the conclusions made? 4g -Pg 12 line 224 -it's not possible to draw this conclusion from the data presented, the p65 blot is too dirty to see the NFkB band in the nuclear fractions.
4h -p-IkBa blot too dirty to draw conclusion. It should be noted that TRIM5 itself stimulates an NFkB signal in 293T cells. The phenotype of the mutants used is inversely correlated with their NFkB activity (via TRIM5 E3 activity). Is this a coincidence or relevant to the phenotype? Figure 6 Generally the blots are overexposed making their interpretation difficult.
For the majority of panels in this figure there is a lack of an IgG control antibody that is important to know that the IPs are specific. Otherwise, the mirroring of input with IP could simply be nonspecific binding of proteins to the protein A agarose etc.
6f -pg14 line 274 -in fact, it looks like the effect of TRIM5 on HDAC1-sp1 binding is weaker in the presence of TNF, and not equivalent as suggested in the text. Figure 7 7j-m -this data appear to be misinterpreted. TRIM5 KO does not rescue the inhibition caused by HDAC1 expression on LTR transcription. Rather, the fold inhibition caused by HDAC1 is equivalent in control or TRIM5 KD cells. In 7 m there seems to be negligible effect caused by HDAC1.
Page 4 line 60 -the sentence is a little ambiguous. The TRIM5 RING is widely known to be an adaptor type E3 for ubiquitination, but not Sumoylation or ISGylation (as in, biochemical evidence, not just overexpressed proteins in cells). The RING fold in general however is involved in all 3 of these. So just to clarify whether this sentence relates to the RING fold in general, or specifically the TRIM5 RING.  Major points: 1

Response:
We thank the reviewer for the helpful comment. As the reviewer suggested, we examined the expression of TRIM5α in the cytoplasmic and nuclear extracts from parental (untransduced) J-Lat cells. The results showed that TRIM5α existed not only in the cytoplasm but also in the nuclear extract of the parental J-Lat cells. Furthermore, we used primary human PBMCs and 293T cells to detect TRIM5α protein distribution. Again, TRIM5α was found in the cytoplasm and nucleus of both PBMCs and 293T cells. Notably, the nuclear extract of PBMCs harbored substantial amounts of TRIM5α, suggesting that TRIM5α may play an important role in PBMCs.
We have added these results in Revised Figure 4h. We also added a description in Lines 232-239, Page 12.

Is the role of TRIM5a exclusive to HIV-1 and viral elements or is this a
general effect also active on host genes? The authors touch upon this issue (line 388-390) but never explore it. In the abstract, the identified mechanism is suggested as "a target for future HIV eradication strategies". This would require the mechanism to predominantly act on non-host elements. In the current study, HIV-1 could easily be seen as a reporter gene regulated by NFkB and SP1. Are also host loci under NFkB control, such as e.g. TNFa, affected by the TRIM5a-HDAC1 interaction? This could be tested by adding a few more primers in the ChIP-PCR experiments (selected parts of Fig 7).

Response:
We thank the reviewer for the constructive suggestion. We performed ChIP-qPCR experiments by using primers for IFNB1 and CXCL10, which are two representative genes under NFkB control. The results showed that TRIM5α-WT but not TRIM5α-ΔRING or TRIM5α-ΔB-box 2 enhanced the recruitment of HDAC1 to the promoters of IFNB1 and CXCL10. Consistently, knockdown of TRIM5α reduced the enrichment of HDAC1 on the promoters of IFNB1 and CXCL10.
We also detected the effect of TRIM5α on the levels of H3K9 acetylation at the 5 promoters of IFNB1 and CXCL10. The results showed that the local histone acetylation levels of these promoters were compromised by overexpression of wild-type TRIM5α but significantly elevated by knockdown of TRIM5α. Please find these results in Revised Figure 8k, i.
Altogether, these data indicate that the effect of TRIM5α is general on NFkB-responsive genes. Thus, we toned down the conclusion that TRIM5α is "a target for future HIV eradication strategies" in the abstract. We have revised the sentence from "These findings provide new insights into the molecular mechanisms by which histone deacetylases are recruited to the retroviral promoters and a target for future HIV eradication strategies" to "These findings provide new insights into the molecular mechanisms by which proviral latency is initially established and activatable proviruses are resilenced by histone deacetylase recruitment"; please see

It is suggested that "TRIM5a suppresses HIV-1 latency reactivation" (line 112).
Instead of presenting a TRIM5a inhibitor as a new LRA, the implications of this mechanism seem to be broader. Given the proposed HDAC-mediated mechanism, TRIM5a recruits HDAC1 to the already active HIV-1 promoter, and subsequently silences it. Apart from TRIM5a having a role in the initial establishment of proviral latency when the provirus is integrated in a chromatin region of acetylated histones, it would also re-silence activatable proviruses which gain acetyl marks during activation. These toggling proviruses poses a main hinder for an HIV-1 cure.

Response:
We thank the reviewer for the helpful comment. We agree with the reviewer that TRIM5a has a role in the initial establishment of proviral latency when the provirus is integrated in a chromatin region of acetylated histones, and it also resilences activatable proviruses that gain acetyl marks during activation. We highlight this conclusion in the abstract as follows: "These findings provide new insights into the molecular mechanisms by which proviral latency is initially established and activatable proviruses are resilenced by histone deacetylase recruitment"; please see Lines 16-18, Page 1.  Regarding the question "Is there any evidence that TRIM5 expression correlates with HIV quiescence in vivo?", we performed some literature research and found that there is one study published in 2021 reporting significantly higher levels of TRIM5α expression in cells from long-term nonprogressors (LTNP) with respect to HIV-1-infected normal progressor patients 1 . This study suggests that TRIM5α may correlate with HIV transcription silencing, yet further investigation is needed.

Major comments:
General: 1. Western blots: • Molecular weight indicators should be displayed on all western blot panels for it to be possible to interpret them.

Response:
We thank the reviewer for the helpful comment. We have now added molecular weight indicators to all western blot panels.
• Exposure -e.g. fig 4h tubulin, it's impossible to interpret as the exposure is too high. This is a recurrent concern.
Response: We thank the reviewer for the helpful comment. We have now replaced the overexposed blots with properly exposed blots for a better interpretation.

Response:
We thank the reviewer for these helpful comments. We apologize for not clearly presenting the construction strategy of the TRIM5 mutants. We have now indicated the domain boundaries in the schematic of TRIM5 mutants in Revised Figure 5a for better clarity.
We agree with the reviewer that domain truncation may cause misfolded and nonfunctional proteins. As the reviewer suggested, we employed single-point mutants B-Box R119E, which interferes with higher-order assembly, and L19R, which disables E2 binding and thus RING activity (we did not use E11R because the ∆RING construct, which was devoid of aa 15-58, impaired the suppression activity of TRIM5alpha, suggesting that E11 does not contribute to TRIM5α inhibition of LTR activation), to determine whether higher-order assembly or RING activity accounts for TRIM5α-mediated LTR regulation.
The Co-IP assay results showed that R119E could indeed significantly impair the interaction of HDAC1 and TRIM5α-WT, as well as the TRIM5α-mediated HDAC1-Sp1 and HDAC1-p50 interactions, while L19R was not able to do so (Revise Figure 7a-c). Moreover, luciferase assays showed that R119E but not L19R greatly reduced the TRIM5α-mediated suppression of LTR-luc, NFKB-luc, and Sp1-luc (Revise Figure 7d). 10 ChIP-qPCR analysis further showed that R119E exhibited impaired ability to enhance the recruitment of HDAC1 to the HIV LTR and decreased impact on the levels of H3K9 acetylation compared to TRIM5α-WT, as shown in Revised Figure 8i Altogether, the findings demonstrated that R119, the previously reported key residue that mediates the higher-order assembly of TRIM5α 2, 3 , is also the main residue that mediates the interaction of HDAC1 and TRIM5α. RING activity, on the other hand, is not related to the interaction between HDAC1 and TRIM5α. The key amino acid(s) in the RING domain that promote the interaction between HDAC1 and TRIM5α have yet to be defined by further endeavors. 11 Again, we deeply appreciate the reviewer for the constructive advice that helps to reveal a more detailed molecular mechanism, which improves the quality of this manuscript. We included figures of these new results in Revised Figures 7 and 8. We have also added a description of these new results in Lines 289-303, Pages 15-16, and Lines 335-343, Pages 17-18.

1d -pg6 line 112 -where is the data showing dose-dependent repression?
Response: We thank the reviewer for pointing this out. We apologize for describing 2 dose treatments as a "dose-dependent" manner, and we have revised the sentence as follows: "Furthermore, we showed that TRIM5α suppressed HIV-1 latency reactivation in the presence of different dosages of TNFα treatment". Please see Lines 112-113, Page 6.
P6 line 120 -Query the interpretation of 1d. Jq1 is the only LRA that loses its phenotype in TRIM5 depleted cells, suggesting that TRIM5 repression and the target of Jq1 are mechanistically linked? Indeed, one model could be that TRIM5 suppresses transcription elongation, rendering JQ1 ineffective in the KD cell. The conclusion in the text 'suggesting that TRIM5 had no effect on transcription elongation' is not supported by the data.

Response:
We thank the reviewer for pointing this out. We agree with the reviewer that JQ1 losing its phenotype in TRIM5-depleted cells suggests that TRIM5 repression and the target of JQ1 are mechanistically linked. It was reported that the mechanism by which JQ1 activates HIV-1 latency is that JQ1 dissociates bromodomain protein Brd4 from the HIV promoter, allowing recruitment of P-TEFb to the HIV LTR 4 . There are also studies reporting that the dissociation of HDAC releases P-TEFb from its inhibitory complex, which leads to the activation of HIV transcription 5, 6 . Therefore, it is possible that JQ1 treatment may saturate the effect of TRIM5α if it is involved in the HDAC-P-TEFb axis. In the present study, we focused on how TRIM5α recruits HDAC1 to p50 and Sp1 and to the HIV-1 LTR. In the near future, we will investigate whether TRIM5α regulates HIV latency in an HDAC-P-TEFb-dependent manner. Therefore, we have revised the text from "suggesting that TRIM5 had no effect on transcription elongation" to "suggesting that the effect of JQ1-mediated activation saturates the effect of TRIM5α"; please see Lines 120-121, Page 6. We also discussed this finding in the Discussion section: "Apart from association with p50 and Sp1,

scenario.
Response: We thank the reviewer for the helpful comment. We agree with the reviewer that these data suggest that these LRAs and TRIM5 work independently to some extent. However, the drugs did not work as effectively as in WT or KD cells, as the fold change in TRIM5KD-mediated latency activation in all LRA-treated cells was less than that in the untreated control (shown below in Rebuttal Figure 2). Hence, we conclude that the targets of these LRAs overlap with the targets of TRIM5.

Figure 3
Switched GFP for Luc -not clear why -so essentially similar data as presented in What is the basis for switching between a stable LTR suppression assay as in

CDK9 and Cyclin T1)? What is the logic for this experiment?
Response: We thank the reviewer for the helpful comment. One possibility to explain "TRIM5 only able to antagonise TNF-induced transcription and not Tat driven elongation" is that TRIM5 may be counteracted by Tat. One study reported that SAMHD1 was able to impair TNFα-induced and PMA-induced HIV-1 reactivation but had no effect on the HIV-1 LTR in the presence of Tat, with an uncharacterized mechanism 7 . Another study reported that the HIV-1 Tat protein interacts with CPSF-73 and counteracts its repressive activity on the HIV-1 LTR promoter 8 . Tat has been reported to disassociate HDAC1 from HIV-1 LTR 9 . Thus, it is possible that Tat abrogates TRIM5α-mediated suppression of the HIV-1 LTR by counterregulating HDAC1 recruitment to the HIV-1 LTR.
In this study, we dissected the mechanism by which TRIM5α suppresses the activation of HIV-1 LTR by TNFα once we did not observe the effect of TRIM5α on 15 Tat-activated LTR-driven expression. We will seek the mechanism by which Tat counteracts TRIM5α-mediated suppression of HIV-1 LTR activation in our next research project. The luciferase activity of the reporters was normalized by the luciferase activity of RL-TK (Renilla luciferase), which was transfected along in each of the reporter assays, to normalize the transfection efficiency. We apologize for the unclearness. We have now added a description of the method of normalization to the text in Lines 164-166, Page 9: "The luciferase activity of the reporters was normalized to the luciferase activity of RL-TK (Renilla luciferase).".

3c-3e -why are the RLU units so different between these experiments?
Response: We thank the reviewer for the comment. A possible reason for the difference in RLU in Figure 3c-3e is that the luciferase activity of Full-LTR is much higher than that of core-LTR. We conducted an experiment that directly compared the luciferase activity of these two constructs, and the results are shown below (Rebuttal letter Figure 4). In addition, the transfection amounts of reporter plasmids and RL-TK may vary between independent experiments, which leads to fluctuations in the final relative luciferase activity units. In 3h-i, there seems to be a TNF-independent TRIM5 phenotype. In 3m-n, there seems to be a TNF-dependent TRIM5 phenotype. Would the authors comment on this? Both assays involved TRIM5 depletion.

Response:
We thank the reviewer for pointing this out. We agree with the reviewer that there is a TNF-independent TRIM5 phenotype in Figure 3h 6. Figure 4 Same concern as above re: clonal effect.

Response:
We thank the reviewer for the helpful comment. As the reviewer suggested, we examined the effect of both TRIM5-KO 10# and 16# on NF-κB-and Sp1-driven gene expression. The new results showed that both clones significantly potentiated the TNF-induced activities of NF-κB-luc and Sp1-luc. Please see Revised Response: We thank the reviewer for the helpful comment. We completely agree with the reviewer that TRIM5 activates the ∆SP1 core LTR presumably because TRIM5 stimulates NFkB expression. We have added this presumption to the sentence; please see Lines 202-205, Page 11: "in the absence of TNFα treatment, TRIM5α suppressed the transcriptional activity of the ΔNF-κB core LTR while activating the ΔSp1 core LTR (Fig. 4a, b), presumably because TRIM5α stimulates NF-κB-luc activity".

4c -we can't see the black bars or any change between conditions, either separate panels or a log panel should be used, although this might alter the conclusions made?
Response: We thank the reviewer for the helpful comment. As the reviewer suggested, we have separated the panel to obtain a better view of the values of each bar. The results showed that TRIM5α was able to activate the 5*κB reporter in the absence of a stimulus, which is consistent with previous reports 10, 11   7. Figure 5 22

It should be noted that TRIM5 itself stimulates an NFkB signal in 293T cells. The phenotype of the mutants used is inversely correlated with their NFkB activity (via TRIM5 E3 activity). Is this a coincidence or relevant to the phenotype?
Response: We thank the reviewer for the helpful comment. The luciferase experiments shown in Figure 5c were performed in the presence of TNFα. Therefore, the ∆RING mutant losing the TRIM5α-WT-mediated suppression of TNF-induced NFkB activity may result from the loss of RING-mediated recruitment of HDAC1 to the p50 mechanism.
8. Figure 6 Generally the blots are overexposed making their interpretation difficult.
For the majority of panels in this figure there is a lack of an IgG control antibody that is important to know that the IPs are specific. Otherwise, the mirroring of input with IP could simply be non-specific binding of proteins to the protein A agarose etc.

Response:
We thank the reviewer for the helpful comment. We apologize for not including the IgG control to distinguish nonspecific binding from specific binding. Therefore, we reperformed the Co-IPs with the IgG control to confirm the TRIM5α-mediated promotion of the interaction between HDAC1 and Sp1 as well as between HDAC1 and p50. The new results showed that there were no nonspecific bands in the IgG control. Please see Revised Figure 6a Thus, we have revised the description from "We also showed that the TRIM5α-mediated HDAC1-Sp1 and HDAC1-p50 interactions persisted in the presence of TNFα (Fig. 6f, g)" to "Notably, the results showed that the TRIM5α-promoted HDAC1-Sp1 and HDAC1-p50 interactions were attenuated by treatment with TNFα (Fig. 6f, g) and that the association of TRIM5α and Sp1 was also markedly reduced upon TNFα treatment (Fig. 6f), suggesting the release of TRIM5α-mediated suppression of Sp1 and NF-κB under latency-reversing stimulation". Please see Lines 281-285, Page 15. We also discussed these results in the Discussion section: "Further studies revealed that TNFα treatment released TRIM5α from the HIV-1 LTR and disrupted TRIM5α's effect on the promotion of the HDAC1-Sp1 and HDAC1-p50 interactions (Fig. 8d, Supplementary Fig. 4 and Fig.   6f, g), which may explain why the effect of TRIM5α on Sp1-luc and NF-κB-luc was exaggerated upon TNFα treatment", please see Line 436-440, Page 22. Figure 7 7j-m -this data appear to be misinterpreted. TRIM5 KO does not rescue the inhibition caused by HDAC1 expression on LTR transcription. Rather, the fold inhibition caused by HDAC1 is equivalent in control or TRIM5 KD cells. In 7 m there 25 seems to be negligible effect caused by HDAC1.

Response:
We thank the reviewer for the helpful comment. We agree with the reviewer that in Figure 7 j-m, TRIM5 KO does not rescue the inhibition caused by HDAC1 expression on LTR transcription, and that in 7 m there seems to be negligible effect caused by HDAC1. We repeated the experiments and obtained similar results.
The fold inhibition by HDAC1 on LTR was calculated and compared between TRIM5 KD cells and control cells in Supplementary Figure 5c, d. Thus, the results indicated that TRIM5 is not fully responsible for the inhibitory effect of HDAC1 on LTR transcription. As HDAC1 has been reported to be regulated by a number of transcription factors, such as YY1, LSF, c-Myc and CTIP2 9, 12, 13 , it is possible that HDAC1 retains efficient inhibition of HIV transcription through those factors.
We have rewritten the description as follows: "However, TRIM5α depletion did not fully abolish the effect of HDAC1 on the repression of HIV-1 full/core LTR-driven transcription ( Supplementary Fig. 5b-d)